The proposed study is designed to elucidate the transport and metabolism of methotrexate and the folate coenzymes in hepatic tissue. A monolyer culture system of rat hepatocytes and a rat hepatoma (H35) cell line will be used to examine the transport processes. A comparative assessment will be made between the normal and transformed cells and the normal cells will be examined to determine the properties that allow these cells to act as the storage site for folates. A transport resistant line of the H35 cells has been developed and the properties of these cells will be compared with the normal H35 cells. Both hepatocytes and especially H35 cells catalyze a rapid conversion of methotrexate to polyglutamate derivatives. The properties of this reaction will be studied in cells in culture and cell free extracts to understand the enzymology and regulation of the reaction. We will also assess the contribution that polyglutamates make to the chemotherapeutic action of methotrexate in H35 cells. Methotrexate bound to macromolecular carriers will be examined to determine their comparative effectiveness against normal and resistant H35 cells.